Nanotechnology-driven tissue engineering strategies are evaluated here for the development of innovative methods aimed at the biological fixation of soft tissue grafts. Specifically, we focus on the challenge of tendon-to-bone integration for Rotator Cuff repair and augmentation. Rotator cuff tear is the most common shoulder injury, with over 75,000 repair procedures performed annually in the US. Our approach to biological fixation centers on the regeneration of the anatomic insertion site between tendon and bone. Given the characteristic spatial variation in cell type, matrix composition and mineral content inherent at the native insertion site, it is expected that interface regeneration will require multiple cell types and a stratified scaffold capable of supporting multi-tissue formation. We have therefore developed a biomimetic, nanofiber-based biphasic scaffold for tendon-bone integration, with each of the phases designed for the formation of the non-mineralized and mineralized regions of the native insertion site. The objective of this proposal is to optimize multi-cell culture and biomimetic scaffold design parameters for interface regeneration and multi-tissue formation. Aim 1 will test the hypothesis that fibroblast and osteoblast response on the nanofiber-based scaffold will be governed by nanofiber geometry and mineral content. Aim 2 will focus on the formation of distinct yet continuous regions of non-calcified and calcified tissue regions on the biphasic scaffold through co-culture of fibroblasts and osteoblasts, as well as the maintenance of these distinct regions in vivo. Our effort to regenerate the anatomic fibrocartilage interface as part of rotator cuff repair represents an innovative solution to a significant clinical challenge. Moreover, the nanofiber-based multiphasic scaffold design and co-culture methods proposed here are highly original. It is anticipated that the successful completion of the proposed studies will facilitate the development of a new generation of integrative fixation devices, as well as demonstrating the potential of nanotechnology for engineering complex musculoskeletal tissue systems that can integrate seamlessly with the body. Biological fixation of the Rotator Cuff tendon grafts to bone poses a significant clinical challenge. This project focuses on the design and optimization of a biomimetic, nanofiber-based scaffold for promoting tendon-to-bone integration post cuff repair, focusing on exercising spatial control of fibroblasts and osteoblasts distribution and multi-tissue formation through multi-phased scaffold design and fibroblast-osteoblast co-culture. Findings from the planned studies will have a significant impact in public health due to the large number of Rotator Cuff repair procedures performed nationally and worldwide. In addition, this project can have broad impact in the translation of tissue engineered grafts to the clinical setting, by enabling the formation of complex tissue systems through graft integration with each other as well as with the host environment.